A striking feature of myeloma cells concerns their tendency to reside in the bone marrow compartment during the main course of the disease evolution. The microenvironment provides the appropriate signals for growth and survival of the tumor cells. Si

A striking feature of myeloma cells concerns their tendency to reside in the bone marrow compartment during the main course of the disease evolution. The microenvironment provides the appropriate signals for growth and survival of the tumor cells. Since small amounts of myeloma cells are also detectable in the peripheral blood, it can be assumed that these cells directly contribute to disease spreading. The detection of these circulating cells implicates that they must be equipped with the appropriate surface molecules that mediate binding to endothelium, responsiveness to chemokines, transendothelial migration and extravasation. It can be assumed that the migration of myeloma cells to the bone marrow is a multistep process as described for normal lymphocytes. The main objective of this research project is to identify molecular mechanisms involved in the homing of myeloma cells. More specifically we aimed to determine: 1) what explains the selective presence of myeloma cells in the bone marrow, 2) which chemotactic signals mediate the migration of myeloma cells and 3) how myeloma cells transmigrate through bone marrow endothelium.

2. RESULTS

1999 & 2000 Brian Novis Research Grants (Initial Research Project)

To answer the question whether the restricted localization of myeloma cells in the bone marrow is the result of selective migration towards bone marrow or/and selective survival in the bone marrow, we performed a study in the in vivo 5T2-mouse MM model. Briefly, the in vivo homing kinetics of 5T2 cells 18 hours after injection were assessed in different organs by tracing radiolabeled cells, by immunostaining of isolated cells and PCR analysis. 5T2 cells were found in bone marrow, spleen and liver with all other organs being negative. Adhesion assays of 5T2MM cells to different types of endothelial cells demonstrated a selective adhesion of 5T2MM cells to bone marrow and liver and not to lung endothelial cells. Therefore we concluded that the specific in vivo localization of the 5TMM cells is a result of the combination of a selective entry/adhesion of the 5T2MM cells in the bone arrow, spleen and liver, and a selective survival and growth of these tumor cells in the bone marrow and spleen but not in the liver (published in British Journal of Cancer, 2000, 82, 953-959) (1). In addition, we could demonstrate in the same model that adhesion to bone marrow endothelial cells and homing to bone marrow of the myeloma cells involves the CD44v10 molecule (published in Cancer Research, 2001, 61, 2862-2865) (2).

The migration of lymphocytes and tumor cells through endothelium is believed to be mediated by chemotactic signals provided by the microenvironment. We demonstrated that laminin-1(LN), a major component of the basement membrane acts as an important chemo-attractant for myeloma cells. This molecule was found to stimulate the in vitro migration of 3 human myeloma cell lines (MM5.1, U266 and MMS.1), as well as murine 5T2MM cells and primary myeloma cells immunomagnetically isolated from patient bone marrow samples. Moreover, we found that human myeloma cell lines and murine 5T2MM cells express the 67 kD laminin receptor (67LR). CD38bright+ plasma cells in fresh myeloma bone marrow samples showed weaker 67LR expression, but expression increased after direct exposure to a bone marrow endothelial cell line (4LHBMC). 67LR has been shown to mediate the actions of LN through binding to CDPGYIGSR, a nine amino acid sequence from the B1 chain of LN. Myeloma cell migration was partially blocked by peptide 11, a synthetic nonapeptide derived from this amino sequence and not by a scrambled (control) peptide. Also a blocking antiserum against 67LR reduced LN-induced migration. Co-injection of peptide 11 with 5T2MM cells in the murine in vivo model of MM resulted in a decreased homing of 5T2MM cells to the bone marrow compartment. We conclude that 67LR on the surface of myeloma cells is involved in the laminin-1 induced migration of myeloma cells and that this mechanism might be important during the extravasation of circulating myeloma cells. (published in British Journal of Cancer, 2001, 85, 1387-1395) (3)

In order to identify additional receptors for chemotactic molecules that can trigger the bone marrow homing of myeloma cells, we started to analyze the functional expression of chemokine-receptors. We could demonstrate by RT-PCR the expression of the chemokine receptor CCR2 in several human myeloma cell lines. FACS analysis revealed CCR2 expression at the surface of all myeloma cell lines and primary myeloma cells, immunomagnetically isolated from bone marrow samples. In addition, we found that the monocyte chemotactic proteins (MCP's) MCP-1, MCP-2 and MCP-3, three chemokines acting as prominent ligands for CCR2, are produced by bone marrow stromal cells. By in vitro migration assays we could demonstrate that myeloma cell lines as well as freshly isolated myeloma cells migrate to MCP-1, to MCP-3 and to a lesser extent to MCP-2. A blocking antibody against CCR2 as well as a combination of antibodies against MCP-1, MCP-2 and MCP-3 significantly reduced the migration of human myeloma cells to conditioned medium of cultured stromal bone marrow cells. These results suggest a potential contribution of CCR2 and the MCP's to the bone marrow homing of human myeloma cells (oral presentation during the 2000 ASH meeting, San Francisco) (4), (manuscript submitted) (5).

As part of this project, we also analyzed the functional role of the motility-related protein-1 (MRP-1/CD9) in myeloma cell migration. Previously, we found by representational difference analysis that MRP-1 transcripts are expressed by the stroma-dependent myeloma cell line MM5.1 and not by the stroma-dependent variant MM5.2. Accordingly, most myeloma cell lines that grow stroma-independently were found to be MRP-1 negative. By analyzing in vitro migration through Transwell filters and Matrigel (basement membrane extract), MM5.2 cells were found to be more motile and invasive than MM5.1 cells. (Acta Oncologica, 2000, 39, 771-776) (6). To test the functional role of MRP-1 in this different migration behavior, we tried to establish MRP-1 expressing transfectants of CD9 negative myeloma cell lines (MM5.2 and MMs-1). These variants were produced by transfection with MRP-1 cDNA (vector provided by Dr. Adachi, Osaka, Japan). However, when the transfectants were compared with the wild type cells no differences in in vitro migration properties could be found. Therefore we could not provide any evidence that the higher motility of MM5.2 cells relates to a differential expression of MCP-1. Interestingly, we found a significantly lower CD9 expression on plasma cells of patients with aggressive myeloma as compared to patients with non-aggressive disease. The recent finding that enhanced CD9 expression on myeloma cells correlates with enhanced susceptibility to cytolysis by IL-2 activated T cells and NK cells, indicates that the observed heterogeneous expression of MCP-1 in myeloma cells relates more to differences in immune-control rather than changes in the migration or motility behavior of the tumor cells. (Manuscript in preparation).

2002 Brian Novis Research Grant (January-August)

Functional role of the metalloproteinases MMP-2 and MMP-9 in the transendothelial invasion of human myeloma cells. With this part of our work we wanted to determine whether myeloma cells are invasive and at which level the transendothelial invasion of human myeloma cells is mediated by the metalloproteinases (MMP's), MMP-2 and MMP-9. Both MMP's have been described to be expressed by cancer cells but their specific role in the extravasation process of myeloma cells is unknown. Using RT-PCR and zymography, we found that MM cell lines and primary MM cells do not expres MMP-2 whereas MMP-9 was expressed in all tested patient samples and 3/8 MM cell lines (8226, LP-1 and MMs1). Furthermore we could demonstrate that MM cells are capable of transendothelial invasion and this process was inhibited in the presence of neutralising MMP-9 antibodies. In addition we demonstrated that human MM cells are more invasive in the presence of endothelial cells as compared to migration through the basement membrane without endothelial cells. We also showed that this endothelial cell-induced stimulation correlates with the up-regulation of MMP-9 in tumor cells, induced by endothelial cells. Final experiments suggests that hepatocyte growth factor ( HGF), produced by the endothelial cells, is involved in this up-regulation. These in vitro experiments demonstrate that human myeloma cells are capable of transendothelial invasion and that the protease MMP-9 plays an important role in this process (oral presentation during the 2002 ASH meeting, Philadelphia) (7).

Functional role of the adhesion molecules CD44, VLA-4 and CD38 in the adhesion to- and the migration through bone marrow endothelium In first instance we showed that human myeloma cell lines (Karpas, LP-1 and 8226) are able to adhere in vitro to bone marrow endothelial cells, but this adhesion is not specific because they can also bind to other endothelial cell types (lung and umbilical cord). In addition we demonstrated that human myeloma celllines as well as primary tumor cells isolated from patient samples, can migrate through transwell filters coated with bone marrow endothelial cells. In order to identify which adhesion molecules are involved in this binding- and migration step, we evaluated the expression of several receptors that are known to mediate interactions of endothelial cells with normal lymphocytes. It was found that human myeloma cell lines express at least three adhesion molecules (i.e. VLA-4, CD44 and CD38) of which the corresponding ligands are expressed by bone marrow endothelium (resp. VCAM-1, hyaluronic acid and CD31). By blocking with VLA-4-, CD38- and CD44- specific antibodies we found that both VLA-4 and CD38 partially (up to 55%) mediate adhesion of myeloma cell lines (Karpas, 8226 and LP-1) to bone marrow endothelial cells while the transendothelial migration was partially inhibited by CD44. These data have to be confirmed with primary isolated myeloma cells.

Functional role of additional chemokine receptors in the transedothelial migration of human myeloma cells. In a previous part of this project (Brian D. Novis Research Grant 2000) we demonstrated that human myeloma cells express the chemokine-receptor CCR2. Moreover we found that this receptor is involved in the migration to the bone marrow micro-environment by interaction with MCP-1, MCP-2 and MCP-3. By ribonuclease protection assay (RPA) and/or Multiplex-PCR, we found that human myeloma cell lines express also transcripts for CCR1,CCR4, CCR8 and CXCR4. In the last part of this project we started to investigate whether these receptors are functionally expressed by myeloma cells and co-act with CCR2 to enhance the selectivity of the tumor cell homing. FACS analysis revealed that human myeloma cell lines show surface expression of CCR1, CCR2 and CXCR4. We could not detect surface expression of CCR4 and CCR8. CCR1, CCR2 and CXCR4 were found to be heterogeneously expressed on plasma cells in a serie of MM bone marrow samples. Currently we are investigating whether this expression correlates with the clinical evolution. In addition we are analysing at which level co-neutralisation of the main ligand-chemokines, MCP-1(2 and 3), MIP-1a and SDF-1a can enhance the inhibition of myeloma cell migration towards bone marrow conditioned medium.